PROJECT SUMMARY Multidrug-resistant (MDR) bacteria are among the most urgent threats to global health, causing life-threatening illnesses and, increasingly, infections for which available antibiotics are simply ineffective. Particularly challenging is resistance against carbapenem antibiotics, an essential clinical barrier against MDR pathogens. Indeed, in 2013, the U.S. Centers for Disease Control and Prevention published a report which designated carbapenem-resistant Enterobacteriaceae (CRE) as an immediate threat to public health that requires urgent and aggressive action. Also concerning is the worldwide emergence of resistance against colistin, a polymyxin antibiotic considered to be a last-line defense against MDR Gram-negative bacteria. Countering the spread of resistance determinants requires a fundamental understanding of the mechanisms affecting their transmission, including in the context of accumulated co-resistance against disparate classes of antibiotics. To address this important gap in scientific knowledge, we propose a forward- looking approach to elucidate a unique bacterial strategy that protects susceptible bacteria from antibiotic killing and promotes the genetic transmission of antimicrobial resistance. Specifically, our investigations will identify the role of outer membrane vesicles (OMVs) produced by CRE in shielding and transforming carbapenem-susceptible bacteria, and define the influence of lipid A modification conferring colistin resistance on promoting these processes. Our innovative proposal will be accomplished by achieving two aims. In Aim 1, we will characterize the biogenesis, lipid A composition, and physiochemical properties of OMVs produced by isogenic colistin-susceptible and -resistant CRE. Protein/lipid quantification, advanced microscopy, immunoassays, and mass spectrometry-based analysis of lipid A will facilitate these studies. In Aim 2, we will determine the effect of colistin resistance on the carriage of bacterial protein and DNA as cargo, and the transmission of carbapenem resistance to susceptible bacteria. Quantitative proteomics, DNA sequencing, and phenotypic assays will form the basis of these investigations. Recent discoveries and preliminary findings by our group of investigators attest to the feasibility of this proposal and provide a complete framework for a) clarifying OMV-mediated transmission of antimicrobial resistance amongst bacteria and b) defining interconnected biological relationships between key resistance determinants. Our proposed research of MDR pathogens will inform infectious diseases epidemiology, prevention, and control programs, as well as the multifaceted functions of OMVs in bacterial pathogenesis and genetic exchange. Thus the current study is highly aligned with the National Institute of Allergy and Infectious Diseases mission to understand, treat, and prevent disease, as well as its mandate to respond to emerging health threats. The focus of the proposed experiments also lends itself to translational avenues of investigation by highlighting novel targets for diagnostic and therapeutic strategies for identifying and treating infections caused by MDR pathogens.